Skeletal osteogenesis is a highly ordered process, involving progressive differentiation and maturation of mesenchymal stem cells which form new bone. Of crucial importance to the bone-forming process is capillary infiltration into the newly forming tissue. In addition to providing nutrients and gas exchange, blood vessels may deliver osteoblastic precursors and/or dictate the spatial orientation of differentiating osteoblasts. In all forms of endochondral osteogenesis, namely embryonic development, ectopic bone formation and fracture repair, osteoblast-mediated bone formation appears to be initiated in a direct relationship to the developing vasculature. The signaling mechanisms by which vascular cells may direct osteogenesis are unknown, but may involve factors secreted from the vascular endothelium, namely endothelin (ET-1), since development of the craniofacial skeleton is abnormal in mice which lack the ET-1 gene. Moreover, preliminary data indicate that ET-1 is also synthesized by osteoblasts, suggesting an autocrine role for ET-1 in bone-forming cells. The objective of the proposed research is to characterize several aspects of osteoblast ET-1 synthesis and responsiveness to support the hypothesis that ET-1 is a novel autocrine mediator of osteogenesis. Specific experimental Aims include: 1) elucidating the pattern of ET-1 and ET receptor expression during osteoblast differentiation; 2) identifying the intrinsic and extrinsic regulatory mechanisms that govern ET-1 and ET receptor synthesis in osteoblasts, as well as osteogenic growth factors that may modulate ET-1 synthesis in vascular endothelial cells; and 3) characterizing the role of autocrine ET-1 production in osteoblast growth and differentiation utilizing ET-1 gene knockout mice as an animal model. Successful outcomes of the proposed research will contribute to our understanding of ET-1 as a growth and differentiation factor, as well as a novel mediator of osteogenesis. The putative role of ET-1 in mediating vascular control of bone formation during embryonic development, growth and fracture repair is a novel consideration in the area of bone research. Furthermore, investigation of the autocrine and/or paracrine role of osteoblast-derived ET-1, aimed at defining the relationships between angiogenesis and bone formation, could improve therapeutic approaches for healing bone fractures, a process which is particularly problematic in aging individuals.